高蛋白酶酶活酱油发酵用米曲霉的复合诱变选育

Multiple mutation breeding of Aspergillus oryzae with high protease activity in soy sauce fermentation

以市售曲精分离菌株米曲霉CS3.03为出发菌株,经UV—DES复合诱变处理,采用酪素平板法及琼脂块法进行初筛,结合Folin—酚法复筛结果对比其初筛效果,低盐固态发酵试验测试高蛋白酶酶活突变株的发酵性能,传代试验测试其遗传稳定性。结果表明:琼脂块法的初筛效率明显高于酪素平板法;对初筛菌株进行制曲复筛,获得5株有生产潜力的高蛋白酶酶活突变株,酱油发酵试验结果证明突变株氨态氮含量、全氮利用率均高于出发菌,其中突变株P7、Y5发酵制得酱油氨态氮含量分别提高14.56%,11.26%,全氮利用率分别提高3.83%,4.09%;遗传稳定性测试证明P7具有良好工业应用价值且遗传稳定性好,其蛋白酶酶活平均为4 994.18U/g干基,比出发菌株提高173.92%。

The original strain of Aspergillus oryzae CS3.03 isolated from koji powder was treated with the composite mutagen UVDES.The mutants were screened with the method of casein plate and agar block.According to re-screening results detecting with the method of Folin-phenol,and the better screening means was found.At the same time,the fermentation ability of high protease activity mutants were tested in the state of low-salt solid-state fermentation technology,and the genetic stability of the best was tested by repeated multiplying.The results showed that the screening efficiency of agar block method was significantly higher than casein plate method.Five mutants of high protease activity with production potential were obtained by re-screening.The result of soy sauce fermentation test proved that the content of ammonia nitrogen and the utilization ratio of total nitrogen were higher than those of the original strain.The ammonia nitrogen in soy sauce fermented by mutant P7 and Y5increased 14.56% and 11.26%respectively,and the utilization ratio of total nitrogen increased 2.92% and 3.12%in the same situation.Genetic stability test showed that P7 had great industrial application value and genetic stability,and its average protease activity was4 994.18U/gdb,which was 173.92% higher than that of the original bacteria.